Protein Summary

The LBA1001 gene from Lactobacillus acidophilus (accession number YP_193882, gi number 58337297) encodes 17 kDa, 150 amino acid long protein that has been annotated as a putative trp repressor binding protein. This annotation was probably based on the sequence similarity between this protein and trp repressor binding proteins (for example FFAS detected 22% sequence identity between this protein and Flavoprotein wrbA, PDB code: 36bi, FFAS score:-47.400). While, 3b6i was originally named tryptophan (W) repressor binding protein (wrbA), it has been shown that 36bi is actually a NAD(P)H:quinone oxidoreductase (reference #1).

LBA1001 belongs to the PFAM Flavoprotein clan (CL0042), which currently contains four families, including best known Flavodoxin Pfam domain (PF00258) (PFAM e.value of 0.001), but it is likely to form a new family in this clan. Flavodoxins are electron-transfer proteins that function in various electron transport systems. They bind one FMN molecule, which serves as a redox-active prosthetic group and are functionally interchangeable with ferredoxins. During the structure determination and refinement, electron density clearly revealed the presence of an FMN prosthetic group. The presence of this FMN prosthetic group and structural similarity with NADH:Quinone Oxidoreductase WrbA from E. coli could perform a similar role as an oxidoreductase in Lactobacillus acidophilus. The biologically-relevant oligomerization state most likely is a monomer. The structure LBA1001 is a classical flavodoxin fold, and is comprised of a central five-strand parallel beta-sheet surrounded by two α-helices on both sides of the sheet.

Ribbon representation of the YP_193882.1 monomer. A tightly bound FMN cofactor is shown as sticks.

During the course of the crystallographic refinement, additional electron density was observed surrounding the sidechain of SeMet 46 (Mse 46) in the vicinity of the FMN cofactor. The relatively enhanced resolution of the refinement strongly suggested that this residue can be modeled as an oxidized SeMet with the modification showing stereoselectivity. The image shown above shows the structure in the vicinity of the oxidized SeMet 46 with 2fo-fc contoured at 1.5 sigma (blue) superimposed on the structure.

A SSM search was performed using the refined structure of YP_193882.1. The top hits from this search are listed:

Least squares superposition of the structures of YP_193882.1 (green) and the solution structure of thetwo-electronreduced flavodoxin from Megasphaera elsdenii (blue)

The most significant structural difference between YP_193882.1 and related structures is the presence polypeptide insertion between residues 35 and residues 68. The conserved sidechains Met 46 and Tyr 47 implicated in a possible catalytic mechanism are found on this loop. This insertion region is likely to be part of a cleft that helps position a potential substrate near FMN for electron transfer.

3) Sequence comparisons

LBA1001/YP_193882 is closely homologous to several proteins from various Lactobacillus species, variously annotated as trp repressors or flavodoxins, but the differences in these annotations reflect different annotation protocols rather then differen functions of these proteins, Top five homologs as identified by NCBI BLAST search are:

The genes surrounding YP_193882.1 have been sequenced . An ATP-DNA dependent helicase and an exonuclease subunit immediately flank the YP_1938832.1. Immediately below is a table of the neighboring genes:

Amino acid sequence alignment for YP_193882.1.1 and the top scoring amino acid sequence sdentity hits from the NCBI BLAST seach. Residues that are strictly conserved include (red) several residues at the N-terminal region. Three residues in the immediate vicinity of the active site sulfur, Tyr 14, Tyr 27, and His 31 are strictly conserved across the homologues. This region at the N-terminus includes some residues that interact with the phosphate moiety on the FMN cofactor in the structure. Tyr 47, a residue implicated to be involved in catalysis and described above in the structure summary is also at the active site.

Ligand Summary

The FMN
cofactor binds at one end of the monomer with the plane of the
isoalloxazine ring on the flavin moiety perpendicular to the long
axis of the monomer. One side of the planar face of the isoalloxazine
ring is partially exposed to solvent. On this solvent exposed face,
the sidechains of Mse 46 and Tyr 47 are nearby. On the opposite side
of the isoalloxazine ring is situated the sidechain of Trp 86 with
the carbonyl oxygen atom of Val 85 within van der Waals contact
distance to the center of the cofactor ring.Thecrystal structure
shows that the sidechain of Tyr46 from a lattice symmetry-related
molecule is within hydrogen bonding distance of the modified Mse 46,
and this Tyr 46 on a lattice symmetry-related molecule is within van
der Waals contact distance of the FMN cofactor. Although
size-exclusion chromatography and packing calculations indicate that
the predominant oligomerization state on YP_193882.1 is a monomer,
the crystal structure at this lattice interface is similar to the
structure at the active site of the E coli structural homolog where the active site is comprised of residues from three different
subunits. In contrast, the structure described here is a monomer in solution. The structure at the interface of the symmetry-related
molecules in crystals of the YP_193882.1 protein near the FMN binding site could have implications for how
FMN interacts with potential redox partners.